Largely
because of deer management strategies and increased forage availability, deer
populations have rapidly increased throughout North
America throughout
the past decades
(Cote et al. 2004). Overabundance has contributed to significant economic losses
in transportation, agriculture, and forestry, as well as to transmission of
disease. Deer damage to
agriculture has been recognized as a substantial economic problem for some time
(Wywialowski 1998). In the Pacific northwestern
United
States, damage to
forest resources by
black-tailed deer is considered a significant impediment to reforestation
efforts (Nolte and Dykzeul 2002). Increased agricultural losses because of deer
browse have also
been reported in Europe (Santilli
et al. 2004). Browse damage can be lethal to plants, while also reducing the
future value of crops via decreased yields and plant deformities
(Nolte 1998). However, browse damage is not limited to commercial agriculture
and reforestation efforts. Overabundant deer may have a significant impact on
plant community structure and ecosystem properties (Cote et al. 2004). High
rates of deer browsing suggest the possible extinction of valuable forest
understory herbs (Mcgraw and Furedi 2005). A number of commercially available
products are marketed to deter browsing of trees and shrubs by deer. These
products contain a broad range of presumed active ingredients—some more
effective than others (Nolte and Wagner 2000). The majority of these products
are contact repellents that must be applied directly onto the plants to be
effective. Among contact repellents, four different modes of action have been
proposed: flavor aversion learning, taste modification, chemical irritation, and
fear (Nolte and Wagner 2000). We recently demonstrated that a number of
methionine containing proteins
minimize browsing by making treated plants less palatable to deer (Kimball and
Nolte 2005). Among these, casein has the potential for commercial use as a deer
epellent. Here, we describe three experiments conducted to evaluate several
protein sources as repellents for protecting conifers from deer browse damage.
We sought to develop a new repellent formulation that effectively minimized
browse damage, was easy to prepare in water, and was relatively inexpensive
versus commercial repellent products.

Forty-eight
captive 1- to 3-year old, black-tailed deer (Odocoileus
hemionus columbianus) were used
for the bioassays conducted in 0.2 ha outdoor pens. The same 24 deer were used
in experiments 1 and 2; whereas 24 different deer were used in experiment 3.
Shelter and ad libitum pelleted basal ration (USDA Deer Pellet; X-Cel Feeds,
Tacoma,
WA), water,
and mineral block were provided throughout each experiment. Naturally occurring
forage in the pens was limited to an assortment of cool season grasses. The
experiments were conducted February 2004 to February 2005 and approved by
the Institutional Animal Care and Use Committee of the
USDANationalWildlifeResearchCenter.

Experiment
1

In each of
eight 0.2-ha pens, western redcedar (Thuja
plicata) saplings
(_60 cm) were
planted in five unique plots consisting of 12 trees per plot arranged in a 3
_
4
arrangement with 1 meter spacing. To assure independence of treatments, plots
were separated by at least 3 meters and treatments were randomly assigned (each
treatment represented by one plot in each pen). Treatments were applied by
spraying individual saplings uniformly with 0.054% (v/v) Tactic solution in tap
water with a tank-type garden sprayer. The wetted saplings were immediately
dusted by hand with the appropriate powdered treatment. The four
methioninecontaining treatments were: egg albumen, CMP, 5% methionine in lime,
and Baker’s soy flour. Lime was included in the experiment as a control. Three
deer were confined to each pen for the duration of the
experiment and provided ad libitum access to test trees for 22 days. The number
of bites on each tree were recorded on days 1, 2, 3, 4, 5, 7, 8, 12, 16, 19, and
22, or until the individual
tree was completely consumed (defined as 25 cumulative bites) according to
previously established procedures(Nolte 1998). Severe browse damage was defined as 10
cumulative bites to an individual tree. Experiment 1was conducted from
Feb. 2 to
27, 2004.

Experiment
2

Experiment 2
was similarly conducted with five plots replicated in eight pens except that
each plot consisted of nine western redcedar saplings in a 3 _
3 array. The
five treatments consisted of three casein-related sources: CMP, EAC, and HC, a
positive control (BGR-P), and a control [0.054% (v/v) Tactic spray only]. Three
deer were confined to each of eight pens for the 16 days of the experiment. The
number of bites on each tree were recorded on days 1, 2, 3, 6, 9, 13, and 16, or
until the individual tree was completely consumed.
Experiment 2 was conducted from May 26 to June 11,
2004.

Experiment
3

The final
experiment was similarly conducted with six plots (12 western redcedar saplings
per plot in 3 _
4 array) in
each of eight 0.2-ha pens. The six treatments used in experiment 3
consisted of four HC solutions, Plantskydd, and a control (Tactic only). For
this experiment, the sticker was mixed with tap water at a concentration of
0.26% (v/v). The four HC
solutions were prepared by adding the powder to the sticker solution to yield HC
concentrations of 2, 4, 8, and 12% (w/v). Treatments were applied to the
saplings by handheld spray bottle. During application, occasional shaking of the
container was required to keep HC suspended in the 8 and 12% solutions. Three
deer were confined to each pen for the duration of the experiment and provided
ad libitum access to test trees for 21 days. The number of bites on each tree
were recorded on days 1, 2, 3, 4, 7, 9, 14, and 21, or until the individual tree
was completely consumed. Experiment 3 was conducted from Jan. 27 to
Feb. 16,
2005.

Statistical
Analyses

For each
experiment, a Kaplan-Meier survival analysis was performed to compare
survivability distribution functions among treatments, using the Wilcoxon test
of equality (PROC
LIFETEST; SAS/STAT 2002, SAS/STAT version 9.1; SAS Institute Inc.,
Cary,
NC). Failure
day was defined as the first experimental day when severe browse (10 cumulative
bites) was measured on an individual tree. Trees that survived to the end of the
experiment (did not meet definition of failure) were assigned a failure day of
25 and censored
according to the standard application of survival analysis. Failure data were
also analyzed by ranking failure day among treatments in each pen (1
_
shortest
failure day) and subjecting the rank data to Kruskal-Wallis analysis (Iman
1982). Rank was the rsponse for the nonparametric analysis with treatment the
fixed effect. Multiple comparisons of mean ranks were made using Fisher’s least
significant difference (LSD option;
SAS/STAT 2002). A separate analysis was conducted for each of the three
experiments.

10
bites) by day 16 of the experiment (Figure 1). Forty percent of trees treated
with CMP and 26% of trees treated with albumen
were not severely browsed by the end of the experiment.
Kruskal-Wallis analysis of the ranked data in experiment 1 demonstrated a
significant treatment effect (P
_
0.0001) with
multiple comparisons of the means indicating that failure day rank followed the
order: CMP _
albumen
_
soy
_
5%
methionine _
lime.
Kruskal-Wallis analysis of the ranked data from experiment 2 demonstrated that
CMP, EAC, HC, and BGR-P were each effective in minimizing browse with respect to
the control (P
_
0.0001). One
hundred percent of trees treated with the
casein sources or BGR-P were protected from browse damage (Figure 2).
Conversely, 71% of control trees were severely browsed by the end of the 16-day
experiment. The results of experiment 3 established that deer avoidance of
HC-treated trees was concentration-dependent (Figure 3). Kruskal-Wallis analysis
of the ranked data demonstrated a significant treatment effect (P
_
0.0001) with
multiple comparisons of the means indicating failure day rank followed the
order: Plantskydd _
12% HC
_
8%
HC
_
4% HC
_
2% HC
_
control.

Discussion

In each
experiment, treatments were visibly apparent as white- or cream-colored powder
adhering to unbrowsed foliage throughout the tests. There was no indication that
plot assignment, or proximity of a treatment plot to another, impacted relative
preference for the treatments. This is consistent with the observation that deer
repellents such as BGR-P and Plantskydd have “aversive distances” of less than 1
meter (Nolte and Wagner 2000). This is the distance from a repellent-treated
food source that deer will avoid an untreated test food. In practice, the
aversive distance of contact repellents is typically 0 meters, wich is why
reapplication is frequently necessary to protect new growth. Therefore, 3 meters
was considered sufficient distance between plots to avoid confounding effects of
treatment interaction in these experiments. The treatments used in experiment 1
were chosen because they each contain methionine. Many proteins do not contain
methionine or are methionine-limited (Friedman 1996). For example, porcine
collagen (gelatin) containslitthionine and was not avoided by deer (Kimball and Nolte 2005). Among
experiment 1 treatments, CMP and albumen contain approximately six times more
protein bound. methionine than Baker’s soy. Previous chemical analyses
demonstrated that methionine was protein-bound (i.e., not present as the free
amino acid) in these sources (Kimball and Nolte 2005). The proteins used in
these experiments all contained less than 5% methionine. Experiment 1 confirmed
that CMP and albumen were more effective repellents than soy flour, which has
low methionine content. More importantly, results of experiment 1 suggest that
proteins with protein-bound methionine were more effective than the free amino
acid. Although Kruskal-Wallis analysis established that the 5% methionine
treatment was more effective than the control (lime only), it was no more
effective in reducing browse damage than the soy treatment that has low levels
of protein-bound methionine. Additionally, consumption of the lime control in
experiment 1 indicated
that avoidance of protein treatments did not result from simple tactile or
visual cues. Experiment 2 was designed to compare the repellency of three casein
sources versus a positive control. CMP contains all protein fractions present in
skim milk (whey and casein). EAC is the casein fraction of skim milk. HC is the
enzymatic digest of casein. Enzymatic hydrolysis yields small peptides and free
amino acids from the intact proteins. BGR-P was chosen as the positive control
for this experiment because it is a powder that could be delivered in the same
manner as the casein treatments. Furthermore, it is a contact repellent with
proven efficacy in bioassays with captive deer (Nolte and Wagner 2000).
Activity of BGR-P is attributed to short-chained aliphatic aldehydes produced by
auto-oxidation of egg lipids (Oita et al. 1977). The results of experiment 2
demonstrated that CMP, EAC, and HC were each as effective as the positive
control. The results
of experiments 1 and 2 indicated that methionine-containing proteins as well as
hydrolysates of methionine-containing proteins are potentially effective deer
repellents
that warrant further investigation. They further suggest that of the numerous
products of casein hydrolysis, the active ingredient(s) are probably small
peptides containing methionine—not methionine present as the free amino acid.
The decision to focus on HC for the development of a new repellent was based on
the assumption that its water solubility would be greater than intact proteins
as a consequence of the hydrolysis process. The choice of HC was further
justified when it was demonstrated that white-tailed deer
(Odocoileus
virginianus) avoided
HC-treated food, but not EAC-treated food in one-choice feeding trials after
food deprivation (Kimball et al. 2005).

Experiment
3 was conducted to determine the effective HC concentration required to
minimize deer browsing. Ready-to-use, premixed Plantskydd was used as the
positive control for
this experiment because the liquid could be applied to the test trees in
identical manner as the HC formulations. Plantskydd (active ingredient dried
blood) has also
exhibited proven efficacy as a contact repellent in bioassays with captive deer
(Nolte and Wagner 2000). The efficacy of the HC treatments was directly
proportional to HC concentration. Plantskydd, 8% HC, and 12% HC all effectively
reduced deer browsing of a preferred conifer species throughout the 3-week
period of the experiment. Browse damage to saplings treated with Plantskydd,
BGR-P, and various casein sources varied among the three experiments. For
example, not a single tree treated with CMP was severely browsed in experiment
2. Conversely, 60% of trees treated with CMP were severely browsed in experiment
1. The motivation of herbivores to use a particular resource is
subject to many variables, including experience with the food, nutritional
state, and food alternatives (Provenza 1995). This is true for deer browsing of
agricultural resources in natural systems as well as the experiments described
here. It is unlikely that the deer’s motivation to browse test trees was
consistent among experiments. Accordingly, the results from each experiment must
be considered independently. Any comparisons among the three experiments can
only be made with respect to the control treatments. These experiments indicate
that HC is an effective repellent for reducing browse damage to forest
resources. Specifically, a liquid formulation consisting of 8% or 12% HC with
0.26% latex-based sticker shows great promise for operational use. Although this
formulation offers no advantages versus commercially available products with
respect to labor investments (it must be delivered to seedlings in the same
manner as the commercial products), potential savings in material costs are
significant. At the time of publication, a 12% HC formulation would cost
approximately $6 USD per 4.0 L in total material costs. Four liters would be
capable of treating _500 30-cm
seedlings. This price is based on the current retail price for the latex sticker
and the cost of HC when purchased in bulk (e.g., 900 Kg pallet). The price per
equivalent volume when HC is purchased in smaller quantities (e.g., 22 Kg bags)
would be approximately $8 USD. The cost of an HC repellent formulation could be
significantly reduced by using an 8% HC formulation, which was found to be as
effective as the 12% formulation in these bioassays. However, because the price
of HC is subject to worldwide milk stores and economics,
the price of a HC repellent formulation could fluctuate proportionally. By
comparison, commercial deer repellent products purchased in bulk or concentrate
typically cost between $15 and $25 USD per equivalent
coverage.